Abstract Sexually transmitted infection by herpes simplex virus type-2 (HSV-2) is a major health problem. HSV-2 genital infections can also significantly enhance susceptibility to human immunodeficiency virus. There is no vaccine or protective therapy available to control the transmission of the virus, which is on the rise. In lieu of an effective vaccine, we recently demonstrated the feasibility of a novel microbivac approach as an effective mucosal immunity boosting virucidal platform. Under this strategy, uniquely designed zinc oxide tetrapod nanoparticles (ZOTEN) provide instant microbicidal benefits to the vaginal surface via high-affinity trapping of invading HSV-2 virions. The trapping renders the virions unable to enter cells of the vaginal epithelium. As an additional advantage of this approach, innate and adaptive immune mechanisms are triggered when the virions captured on ZOTEN are presented to and processed by professional phagocytes including mucosal antigen presenting cells (APCs). We also demonstrated that ZOTEN provides adjuvant-like benefits that are very similar to those seen with alum. This proposal will test our hypothesis that inhibition of HSV-2 attachment receptor, heparan sulfate, from cells on the surface of the genital epithelium will enhance the microbivac efficacy of ZOTEN. We propose that inhibition of virus binding to cells will generate stronger infection blockade and a more robust adaptive immune response against the trapped virus. Our hypothesis is supported by brand new data that binding of anti-HSV peptide, G2, to heparan sulfate proteoglycans stops infection and in addition, triggers internalization of heparan sulfate from the surface of cells. Using a mouse model of female genital infection our central hypothesis will be tested by two specific Aims. Aim 1 will test the first part of our hypothesis that inhibition of heparan sulfate by G2 will protect the cells on the vaginal surface from the deleterious effects of HSV-2 while enhancing the virucidal benefits of ZOTEN. Aim 2 will test the hypothesis that in the presence of G2, the ZOTEN/HSV-2 complexes drive stronger adaptive immune responses. We also propose that dual action by the microbivac will generate strong immunotherapeutic effects, which will be confirmed by a second round of HSV-2 infection. Analyses of key cytokines/chemokines will be made to determine the status of mucosal innate immunity under various treatment conditions. Overall, our exploratory and proof-of-concept studies will establish a role for heparan sulfate inhibition in generating higher microbivac efficacy, and guide future designs for more effective prevention of genital herpes transmission and associated disease manifestations.